[Effect of miRNA from Glycyrrhiza uralensis decoction on gene expression of human immune cells].

MicroRNAs(miRNA) are small non-coding RNAs that regulate the expression of protein coding genes by repressing translation of protein coding mRNA or enhancing mRNA degradation. Its functions have attracted more and more attention from the public. In recent years, the cross-border regulation of miRNA has become a new research direction, and provides a new perspective for people to comprehensively understand the functions of miRNA. Plant miRNA is usually methylated and not easy to degrade. According to our previous researches, there were abundant small RNAs in the decoction of dried liquorice, which provides a new way to study the mechanism of action of licorice. In this study, small RNAs extracted from Glycyrrhiza uralensis decoction and synthesized miRNA mimics were used to treat peripheral blood mononuclear cells(PBMC) isolated from healthy volunteers. The gene expression of toll-like receptors(TLRs), some transcription factors, signal molecules and cytokines were analyzed by RT-PCR. The results showed that glycyrrhiza miRNA could significantly regulate PBMC by inhibiting the expression of genes involved in T cell differentiation, inflammation and apoptosis. The study brought new ideas to us in comprehensively studying the mechanism of licorice and developing the traditional Chinese medicine.

Salvia miltiorrhiza polysaccharide activates T Lymphocytes of cancer patients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza polysaccharide (SMP) is one of the most important components in the water extract of Salvia miltiorrhiza Bunge, which has been mainly applied for the prevention or treatment of ischemic encephalopathy and cardiac diseases including myocardial infarction and coronary heart diseases in clinical practice. AIM OF THE STUDY: Our object is to investigate the immune regulation effects of SMP, specifically on the proliferation and cytotoxicity of T lymphocytes through MAPK and NF-κB pathway in peripheral blood of cancer patients. MATERIALS AND METHODS: SMP was prepared through refluxing with ethanol, refluxing with water, Sevage treatment and ethanol precipitation. The lymphocytes were obtained from the peripheral blood of cancer patients. The effect of SMP on T lymphocyte proliferation was investigated by cell counting and flow cytometry. The effect of SMP on the proliferation of cancer cell lines A549, hepG2 and HCT116 was examined by MTT assay. The cytotoxic activity of T lymphocytes treated with SMP was detected by Calcein-acetoxymethyl (Calcein-AM) release. The gene expression of IL-4, IL-6, IFN-γ and toll like receptors (TLRs) was detected by semi-quantitative PCR. The protein expression of mitogen activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway were detected by western blotting. To further verify whether SMP functions through the indicated pathways,, T lymphocytes were treated with SMP and an extracellular regulated protein kinase (ERK) inhibitor (U0126), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) or an inhibitor of NF-κB inhibitor-α (IκBα) (BAY11-7082), respectively. After 24 h co-treatment, the expressions of p-JNK, p-ERK, IκBα, inhibitory kappa B kinase α (IKKα) and inhibitory kappa B kinase ß (IKKß) protein were detected by western blotting, meanwhile cell numbers of T lymphocytes after inhibition were calculated again by cell counter. RESULTS: SMP dose-dependently promoted the proliferation of T lymphocytes of the cancer patients and significantly improved the cytotoxicity of T lymphocytes against cancer cells. However, SMP showed no effect on the proliferation of the tumor cells from the same source. Furthermore, the gene expression of cytokines including IL-4, IL-6 and IFN-γ were also up-regulated. Moreover, SMP enhanced gene expression of TLR1, TLR2 and TLR4; elevated protein expression of p-JNK and p-ERK; increased protein expression of IKKα, and IKKß and decreased IκBα levels. Meanwhile, knockdown of ERK、JNK or IκBα expression with specific inhibitor significantly depressed the proliferation of T lymphocytes treated with SMP, corroborating the specific regulation effect of SMP on T lymphocytes through MAPK and NF-κB signaling pathways. CONCLUSION: SMP specifically promotes the proliferation and enhances cytotoxicity of T lymphocytes in peripheral blood of cancer patients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.

[Extraction of miRNA from Glycyrrhiza uralensis Decoction and Its Effect on Immune Cells].

OBJECTIVE: To extract microRNA(miRNA) from Glycyrrhiza uralensis(liquorice) decoction and to explore its effect on mmune cells. METHODS: With dried processed liquorice, the water decoction was prepared according to the conventional method and subsequently concentrated by rotary evaporation. The concentrated decoction was further freeze-dried by freeze dryer, and miRNAs were extracted with Plant MicroRNA Extraction Kit. The extracted miRNA was digested by DNase I and then analyzed through the agarose gell electrophoresis. The PBMC was isolated from healthy volunteers and treated respectively by liquorice water extract, glycyrrhizic acid, glycyrrhetic acid and liquorice miRNAs. After 24 hours, the cells numbers were counted, and the changes of cell morphology were observed. The expression of CD3, CD56 and HLA-DR were analyzed by flow cytometry to identify the change of cell subsets in PBMC. RESULTS: miRNAs could be extracted from the decoction of dried liquorice which further confirmed the stability of miRNAs. The in vitro culture experiment showed that,compared with the controls, PBMC treated with liquorice miRNAs appeared apparent cell aggregation and increased cell number and HLA-DR+ cells proportion. CONCLUSION: The miRNAs are successfully extracted from the freeze-dried decoction of dried liquorice. It is indicated that liquorice miRNAs have significant stimulative effects on the growth of PBMC and HLA-DR+ cells subset.